EP2061889B1 - Augmentation des niveaux d'alcaloides nicotiniques - Google Patents

Augmentation des niveaux d'alcaloides nicotiniques Download PDF

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EP2061889B1
EP2061889B1 EP06848676.0A EP06848676A EP2061889B1 EP 2061889 B1 EP2061889 B1 EP 2061889B1 EP 06848676 A EP06848676 A EP 06848676A EP 2061889 B1 EP2061889 B1 EP 2061889B1
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nicotine
plant
nicotiana
tobacco
nbb1
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EP2061889A2 (fr
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Takashi Hashimoto
M. Kajikawa
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22nd Century Limited LLC
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22nd Century Limited LLC
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Priority to PL11187201T priority Critical patent/PL2450446T3/pl
Priority to EP19177939.6A priority patent/EP3578661A1/fr
Priority to EP20110187201 priority patent/EP2450446B1/fr
Priority to EP14163682.9A priority patent/EP2792750B1/fr
Priority to PL14163682T priority patent/PL2792750T3/pl
Application filed by 22nd Century Limited LLC filed Critical 22nd Century Limited LLC
Priority to PL06848676T priority patent/PL2061889T3/pl
Publication of EP2061889A2 publication Critical patent/EP2061889A2/fr
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Priority to HK15103733.9A priority patent/HK1203545A1/xx
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • A61P25/34—Tobacco-abuse
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • encoding and coding refer to the process by which a gene, through the mechanisms of transcription and translation, provides information to a cell from which a series of amino acids can be assembled into a specific amino acid sequence to produce an active enzyme. Because of the degeneracy of the genetic code, certain base changes in DNA sequence do not change the amino acid sequence of a protein. It is therefore understood that modifications in the DNA sequences encoding A622 and NBB1, respectively, which do not substantially affect the functional properties of either enzyme are contemplated.
  • the present invention encompasses both methodology and constructs for increasing nicotinic alkaloid content in a Nicotiana plant, by overexpressing A622. Overexpressing both A622 and NBB1 further increases nicotinic alkaloids levels in a Nicotiana plant.
  • expression denotes the production of the protein product encoded by a nucleotide sequence.
  • “Overexpression” refers to the production of a protein product in a transgenic organism that exceeds levels of production in a normal or non-genetically engineered organism.
  • nucleotide sequences are denoted by italicized font (e.g. PMT ), whereas polypeptide sequences are not italicized (e.g. PMT)
  • NIC1 and NIC2 loci are two independent genetic loci in N. tabacum, formerly designated as A and B. Mutations nic1 and nic2 reduce expression levels of nicotine biosynthesis enzymes and nicotine content, generally the nicotine content of wild type > homozygous nic2 > homozygous nic1 > homoyzgous nic1 and homozygous nic2 plants. Legg & Collins, Can. J. Cyto. 13: 287 (1971 ); Hibi et al., Plant Cell 6: 723-35 (1994 ); Reed & Jelesko, Plant Science 167: 1123 (2004 ). In this description, "nic1nic2" denotes tobacco genotypes that are homozygous for both the nic1 and the nic2 mutations.
  • A622 expression refers to biosynthesis of a gene product encoded by SEQ ID NO: 3.
  • A622 overexpression denotes an increasing of A622 expression.
  • A622 overexpression affects an increase in nicotinic alkaloid content for a plant or cell in which the overexpression occurs.
  • A622 overexpression includes the biosynthesis of a gene product encoded by the following: full-length A622 nucleic acid sequence disclosed in Hibi et al. (1994), supra (SEQ ID NO: 3), SEQ ID NO: 3B, and all A622 polynucleotide variants.
  • NBB1 The nucleic acid sequence of NBB1 (SEQ ID NO: 1) has been determined and encodes the polypeptide sequence set forth in SEQ ID NO: 2.
  • NBB1 expression refers to biosynthesis of a gene product encoded by SEQ ID NO: 1.
  • NBB1 overexpression denotes an increasing of NBB1 expression.
  • NBB1 overexpression affects an increase in nicotinic alkaloid content for a plant or cell in which the overexpression occurs.
  • NBB1 overexpression includes biosynthesis of a gene product encoded by the following: SEQ ID NO: 1, SEQ ID NO: 1B, and all NBB1 polynucleotide variants.
  • N. tabacum contains five expressed PMT genes and N. sylvestris contains three expressed PMT genes.
  • Hashimoto et al. Plant Mol. Biol. 37: 25-37 (1998 ); Reichers & Timko, Plant Mol. Biol. 41: 387-401 (1999 ).
  • the PMT gene from N. tabacum was overexpressed in Duboisia hairy root cultures, the levels of nicotine, hyoscyamine, and scopolamine did not increase significantly.
  • Moyano et al. Phytochemistry 59, 697-702 (2002 ).
  • sylvestris had the highest dry weight content of total alkaloids (the sum of nicotine, nornicotine, anabasine and anatabine) at 29,600 ug/g or 2.96 percent, N alata contained the lowest at 20 ug/g or 0.002 percent.
  • the ratio of nicotine to total alkaloid in the leaves of N sylvestris was about 80 percent versus about 95 percent for N tabacum L. Id.
  • the ratio of nornicotine to total alkaloid in N sylvestris leaves was 19.1 percent versus 3 percent for N tabacum L. Id. Based on these large variations among the sixty Nicotiana species, Saitoh et ai. conclude,. "The amount and ratio of total and individual alkaloids present in a plant depend on the species. No clear-cut correlation between alkaloid pattern and classification of the genus Nicotiana seems to exist.” Id. at page 477.
  • the present specification describes methodology and constructs for increasing nicotinic alkaloids in N. tabacum by overexpressing PMT.
  • QPT expression refers to biosynthesis of a gene product encoded by SEQ ID NO: 5.
  • QPT overexpression denotes an increasing of QPT expression.
  • QPT overexpression affects an increase in nicotinic alkaloid content for a plant or cell in which the overexpression occurs.
  • QPT overexpression includes the biosynthesis of a gene product encoded by the following: full-length QPT nucleic acid sequence disclosed in U.S. Patent No. 6,423,520 (SEQ ID NO: 5), SEQ ID NO: 5B, and all QPT polynucleotide variants.
  • nucleic acid construct comprising at least two of A622 , NBB1, QPT, and PMT is introduced into a Nicotiana plant cell.
  • An illustrative nucleic acid construct of the present invention may comprise both QPT and A622.
  • a genetically engineered plant overexpressing QPT and A622 may be produced by crossing a transgenic plant overexpressing QPT with a transgenic plant overexpressing A622. Following successive rounds of crossing and selection, a genetically engineered plant having overexpressing QPT and A622 can be selected.
  • the present invention provides a means for correcting the "negative correlation" between yield and nicotine content in Nicotiana plants by overexpressing a gene encoding a nicotine biosynthesis enzyme in a high-yielding Nicotiana plant.
  • nicotine biosynthesis enzymes which include but are not limited to QPTase, PMTase, A622, NBB1, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), and S-adenosyl-methionine synthetase (SAMS).
  • Increased-nicotine plants resulting there from are then crossed with any desirable commercially acceptable genetic background that maintains high yield.
  • Suitable high-yield Nicotiana plants include but are not limited to Nicotiana tabacum cultivars K 326, NC71, NC72 and RG81. Following successive rounds of crossing and selection, a genetically engineered plant having increased nicotine and increased yield is accordingly produced.
  • the present specification describes producing a plant having increased yield and increased nicotine by overexpressing a gene encoding a nicotine biosynthesis enzyme, such as QPT, PMT, A622, or NBB1, and overexpressing an increased yield gene, such as P Rms, fructose-1 ,6-/sedoheptulose-1, 7 -bisphosphatase, fructose-1,6-bisphosphatase, and sedoheptulose-1, 7 -bisphosphatase, sedoheptulose-1, 7-bisphosphatase in the same plant or cell.
  • a nicotine biosynthesis enzyme such as QPT, PMT, A622, or NBB1
  • an increased yield gene such as P Rms, fructose-1 ,6-/sedoheptulose-1, 7 -bisphosphatase, fructose-1,6-bisphosphatase, and sedoheptulose-1, 7 -bisphosphatase, sedoheptulose-1, 7-bisphosphatas
  • A622 and NBB1 can be introduced into a non-nicotine producing plant or cell, thereby producing nicotine or related compounds in an organism or cell that does not produce these compounds otherwise.
  • a variety of products can be produced from these engineered organisms and cells, including nicotine, nicotine analogs, and nicotine biosynthesis enzymes.
  • non-nicotine producing plant refers to any plant that does not produce nicotine or related nicotinic alkaloids.
  • Illustrative non-nicotine producing plants include but are not limited to Atropa belladonna and Arabidopsis thaliana.
  • a “nicotine analog” has the basic structure of nicotine but may, for example, have different ring substituents.
  • a nicotine analog may substitute a hydrogen (-H) for the methyl group (-CH 3 ) thereby producing nornicotine, which is an analog of nicotine.
  • nicotine analogs may provide similar physiological effects.
  • Cotinine for example, has been cited for its positive effects on improving concentration and memory and, accordingly, is a nicotine analog. Accordingly, nicotine analogs are defined broadly to cover any and all compounds having similar structural and functional activity to nicotine.
  • a nicotinic alkaloid analog can be produced by providing a nicotine analog precursor in a cell culture system.
  • Nicotinic alkaloid biosynthesis genes have been identified in several plant species, exemplified by Nicotiana plants. Accordingly, the present description embraces any nucleic acid, gene, polynucleotide, DNA, RNA, mRNA, or cDNA molecule that is isolated from the genome of a plant species, or produced synthetically, that increases Nicotiana nicotinic alkaloid biosynthesis. Additionally, expression of such nicotinic alkaloid biosynthesis sequence produces nicotinic alkaloids in a non-nicotine producing cell, such as an insect cell.
  • the DNA or RNA may be double-stranded or single-stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also called the anti-sense strand.
  • NBB1, A622, QPT, and PMT include the sequences set forth in SEQ ID NO: 1, 1B, 3, 3B, 5, 5B, 7, and 7B, respectively, as well as nucleic acid molecules comprised of variants of SEQ ID NO: 1, 1B, 3, 3B, 5, 5B, 7, and 7B, with one or more bases deleted, substituted, inserted, or added, which variant codes for a polypeptide with nicotinic alkaloid biosynthesis activity. Accordingly, sequences having "base sequences with one or more bases deleted, substituted, inserted, or added" retain physiological activity even when the encoded amino acid sequence has one or more amino acids substituted, deleted, inserted, or added.
  • A622, NBB1, QPTase, and PMTase may exist, which may be due to post-translational modification of a gene product, or to multiple forms of the respective PMT, QPT, A622, or NBB1 genes.
  • Nucleotide sequences that have such modifications and that code for a nicotinic alkaloid biosynthesis enzyme are included within the scope of the present invention.
  • the poly A tail or 5'- or 3'-end, nontranslation regions may be deleted, and bases may be deleted to the extent that amino acids are deleted. Bases may also be substituted, as long as no frame shift results. Bases also may be "added” to the extent that amino acids are added. It is essential, however, that any such modification does not result in the loss of nicotinic alkaloid biosynthesis enzyme activity.
  • a modified DNA in this context can be obtained by modifying the DNA base sequences of the invention so that amino acids at specific sites are substituted, deleted, inserted, or added by site-specific mutagenesis, for example. Zoller & Smith, Nucleic Acid Res. 10: 6487-500 (1982 ).
  • isolated nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment.
  • recombinant DNA molecules contained in a DNA construct are considered isolated for the purposes of the present invention.
  • Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or DNA molecules that are purified, partially or substantially, in solution.
  • Isolated RNA molecules include in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules, according to the present invention, further include such molecules produced synthetically.
  • Exogenous nucleic acid refers to a nucleic acid, DNA or RNA, which has been introduced into a cell (or the cell's ancestor) through the efforts of humans. Such exogenous nucleic acid may be a copy of a sequence which is naturally found in the cell into which it was introduced, or fragments thereof.
  • endogenous nucleic acid refers to a nucleic acid, gene, polynucleotide, DNA, RNA, mRNA, or cDNA molecule that is present in the genome of a plant or organism that is to be genetically engineered.
  • An endogenous sequence is "native" to, i.e., indigenous to, the plant or organism that is to be genetically engineered.
  • Heterologous nucleic acid refers to a nucleic acid, DNA or RNA, which has been introduced into a cell (or the cell's ancestor) which is not a copy of a sequence naturally found in the cell into which it is introduced.
  • Such heterologous nucleic acid may comprise segments that are a copy of a sequence which is naturally found in the cell into which it has been introduced, or fragments thereof.
  • a "chimeric nucleic acid” comprises a coding sequence or fragment thereof linked to a transcription initiation region that is different from the transcription initiation region with which it is associated in cells in which the coding sequence occurs naturally.
  • nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer, such as the Model 373 from Applied Biosystems, Inc. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 95% identical, more typically at least about 96% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
  • a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence may be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
  • hybridized nucleotides are those that are detected using 1 ng of a radiolabeled probe having a specific radioactivity of 10,000 cpm/ng, where the hybridized nucleotides are clearly visible following exposure to X-ray film at -70 °C for no more than 72 hours.
  • Differences between two nucleic acid sequences may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • nucleic acid molecule is at least 95%, 96%, 97%, 98% or 99% identical to a reference nucleotide sequence refers to a comparison made between two molecules using standard algorithms well known in the art and can be determined conventionally using publicly available computer programs such as the BLASTN algorithm. See Altschul et al., Nucleic Acids Res. 25: 3389-402 (1997 ).
  • nucleic acid molecules comprising the nucleotide sequence of SEQ ID NOs.: 1, 1B, 3, 3B, 5, 5B, 7, and 7B, respectively, which encode an active nicotine biosynthesis enzyme, wherein the enzyme has amino acid sequence that corresponds to SEQ ID NO.: 2, 4, 6, and 8, respectively, and wherein the protein of the invention encompasses amino acid substitutions, additions and deletions that do not alter the function of the nicotine biosynthesis enzyme.
  • a “variant” is a nucleotide or amino acid sequence that deviates from the standard, or given, nucleotide or amino acid sequence of a particular gene or protein.
  • the terms “isoform,” “isotype,” and “analog” also refer to “variant” forms of a nucleotide or an amino acid sequence.
  • An amino acid sequence that is altered by the addition, removal, or substitution of one or more amino acids, or a change in nucleotide sequence, may be considered a “variant” sequence.
  • the variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine.
  • a variant may have "nonconservative" changes, e.g., replacement of a glycine with a tryptophan.
  • Analogous minor variations may also include amino acid deletions or insertions, or both.
  • Guidance in determining which amino acid residues may be substituted, inserted, or deleted may be found using computer programs well known in the art such as Vector NT! Suite (InforMax, MD) software.
  • Vector NT! Suite InforMax, MD
  • “Variant” may also refer to a "shuffled gene” such as those described in Maxygen-assigned patents.
  • a sequence that increases nicotinic alkaloid biosynthesis is incorporated into a nucleic acid construct that is suitable for plant or cell transformation.
  • a nucleic acid construct can be used to overexpress A622 ,and also optionally additionally at least one of NBB1 , PMT, and QPT in a plant, as well as express A622 and NBB1 , for example, in a non-nicotine producing cell.
  • Recombinant nucleic acid constructs may be made using standard techniques.
  • the DNA sequence for transcription may be obtained by treating a vector containing said sequence with restriction enzymes to cut out the appropriate segment.
  • the DNA equence for transcription may also be generated by annealing and ligating synthetic oligonucleotides or by using synthetic oligonucleotides in a polymerase chain reaction (PCR) to give suitable restriction sites at each end.
  • PCR polymerase chain reaction
  • the DNA sequence then is cloned into a vector containing suitable regulatory elements, such as upstream promoter and downstream terminator sequences.
  • nucleic acid constructs wherein a nicotinic alkaloid biosynthesis-encoding sequence is operably linked to one or more regulatory sequences, which drive expression of the nicotinic alkaloid biosynthesis-encoding sequence in certain cell types, organs, or tissues without unduly affecting normal development or physiology.
  • Promoter connotes a region of DNA upstream from the start of transcription that is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • a "constitutive promoter” is one that is active throughout the life of the plant and under most environmental conditions. Tissue-specific, tissue-preferred, cell type-specific, and inducible promoters constitute the class of "non-constitutive promoters.”
  • “Operably linked” refers to a functional linkage between a promoter and a second sequence, where the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. In general, “operably linked” means that the nucleic acid sequences being linked are contiguous.
  • Promoters useful for expression of a nucleic acid sequence introduced into a cell to increase expression of A622, NBB1, PMTase, or QPTase may be constitutive promoters, such as the cauliflower mosaic virus (CaMV) 35S promoter, or tissue-specific, tissue-preferred, cell type-specific, and inducible promoters.
  • CaMV cauliflower mosaic virus
  • Preferred promoters include promoters which are active in root tissues, such as the tobacco RB7promoter ( Hsu et al. Pestic. Sci. 44: 9-19 (1995 ); U. S. patent No.
  • the vectors may also contain termination sequences, which are positioned downstream of the nucleic acid molecules, such that transcription of mRNA is terminated, and polyA sequences added. Exemplary of such terminators are the cauliflower mosaic virus (CaMV) 35S terminator and the nopaline synthase gene (Tnos) terminator.
  • the expression vector also may contain enhancers, start codons, splicing signal sequences, and targeting sequences.
  • Expression vectors may also contain a selection marker by which transformed cells can be identified in culture.
  • the marker may be associated with the heterologous nucleic acid molecule, i.e., the gene operably linked to a promoter.
  • the term "marker” refers to a gene encoding a trait or a phenotype that permits the selection of, or the screening for, a plant or cell containing the marker. In plants, for example, the marker gene will encode antibiotic or herbicide resistance. This allows for selection of transformed cells from among cells that are not transformed or transfected.
  • Suitable selectable markers include adenosine deaminase, dihydrofolate reductase, hygromycin-B-phosphotransferase, thymidne kinase, xanthine-guanine phospho-ribosyltransferase, glyphosate and glufosinate resistance, and aminoglycoside 3'-O-phosphotranserase (kanamycin, neomycin and G418 resistance). These markers may include resistance to G418, hygromycin, bleomycin, kanamycin, and gentamicin.
  • the construct may also contain the selectable marker gene Bar that confers resistance to herbicidal phosphinothricin analogs like ammonium gluphosinate. Thompson et al., EMBO J. 9: 2519-23 (1987 ). Other suitable selection markers are known as well.
  • Visible markers such as green florescent protein (GFP) may be used.
  • GFP green florescent protein
  • Replication sequences may also be included to allow the vector to be cloned in a bacterial or phage host.
  • a broad host range prokaryotic origin of replication is used.
  • a selectable marker for bacteria may be included to allow selection of bacterial cells bearing the desired construct. Suitable prokaryotic selectable markers also include resistance to antibiotics such as kanamycin or tetracycline.
  • nucleic acid sequences encoding additional functions may also be present in the vector, as is known in the art.
  • T-DNA sequences may be included to facilitate the subsequent transfer to and incorporation into plant chromosomes.
  • the present invention comprehends the genetic manipulation of a Nicotiana plant for increasing nicotinic alkaloid synthesis via introducing a polynucleotide sequence that encodes an enzyme in the pathway for nicotinic alkaloid synthesis. Additionally, the disclosure provides methods for producing nicotinic alkaloids and related compounds in non-nicotine producing plants, such as Arabidopsis thaliana and Atropa belladonna.
  • Genetically engineered encompasses any methodology for introducing a nucleic acid or specific mutation into a host organism.
  • a tobacco plant is genetically engineered when it is transformed with a polynucleotide sequence that increases expression of a gene, such as A622 or NBB1 , and thereby increases nicotine levels.
  • a tobacco plant that is not transformed with a polynucleotide sequence is a control plant and is referred to as a "non-transformed" plant.
  • the "genetically engineered” category includes “transgenic” plants and cells (see definition, infra), as well as plants and cells produced by means of targeted mutagenesis effected, for example, through the use of chimeric RNAIDNA oligonucleotides, as described by Beetham et al., Proc. Nat 'l. Acad. Sci. USA 96: 8774-8778 (1999 ) and Zhu et al., loco cit. at 8768-8773, or so-called "recombinagenic olionucleobases,” as described in PCT application WO 03/013226 .
  • a genetically engineered plant or cell may be produced by the introduction of a modified virus, which, in turn, causes a genetic modification in the host, with results similar to those produced in a transgenic plant, as described herein. See, e.g., U.S. patent No. 4,407,956 . Additionally, a genetically engineered plant or cell may be the product of any native approach (i.e., involving no foreign nucleotide sequences), implemented by introducing only nucleic acid sequences derived from the host species or from a sexually compatible species. See, e.g ., U.S. published application No. 2004/0107455 .
  • Plant is a term that encompasses whole plants, plant organs ( e. g . leaves, stems, roots, etc.), seeds, differentiated or undifferentiated plant cells, and progeny of the same.
  • Plant material includes, without limitation, seeds suspension cultures, embryos, meristematic regions, callus tissues, leaves, roots, shoots, stems, fruit, gametophytes, sporophytes, pollen, and microspores.
  • the class of plants which can be used is generally as broad as the class of higher plants amenable to genetic engineering techniques, including both monocotyledonous and dicotyledonous plants, as well as gymnosperms.
  • a preferred nicotine-producing plant includes Nicotiana, Duboisia, Anthocericis and Salpiglessis genera in the Solanaceae or the Eclipta and Zinnia genera in the Compositae.
  • Tobacco refers to any plant in the Nicotiana genus that produces nicotinic alkaloids. Tobacco also refers to products comprising material produced by a Nicotiana plant, and therefore includes, for example, expanded tobacco, reconstituted tobacco, cigarettes, cigars, chewing tobacco or forms of smokeless tobacco, snuff and snus made from GE increased-nicotine tobacco. Examples of Nicotiana species include but are not limited to the following: Nicotiana acaulis, Nicotiana acuminata, Nicotiana acuminata var.
  • Nicotiana otophora Nicotiana paniculata, Nicotiana pauciflora, Nicotiana petunioides, Nicotiana plumbaginifolia, Nicotiana quadrivalvis, Nicotiana raimondii, Nicotiana repanda, Nicotiana rosulata, Nicotiana rosulata subsp. ingulba, Nicotiana rotundifolia, Nicotiana rustica, Nicotiana setchellii, Nicotiana simulans, Nicotiana solanifolia, Nicotiana spegazzinii, Nicotiana stocktonii, Nicotiana suaveolens, Nicotiana sylvestris, N.
  • tobacco hairy roots refers to tobacco roots that have T-DNA from an Ri plasmid of Agrobacterium rhizogenes integrated in the genome and grow in culture without supplementation of auxin and other phytohormones. Tobacco hairy roots produce nicotinic alkaloids as roots of a tobacco plant do. These types of roots are characterized by fast growth, frequent branching, plagiotropism, and the ability to synthesize the same compounds as the roots of the intact plant. David et al., Biotechnology 2: 73-76.(1984 ). Roots of Solanaceae plants are the main site of tropane alkaloid biosynthesis, and hence hairy root cultures also are capable of accumulating high levels of these metabolites.
  • Non-nicotine producing cells refer to a cell from any organism that does not produce nicotine.
  • Illustrative cells include but are not limited to plant cells, such as Atropa belladonna, Arabidopsis thaliana, as well as insect, mammalian, yeast, fungal, algal, or bacterial cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990 ).
  • insect cell refers to any insect cell that can be transformed with a gene encoding a nicotine biosynthesis enzyme and is capable of expressing in recoverable amounts the enzyme or its products.
  • Illustrative insect cells include Sf9 cells (ATCC CRL 1711).
  • “Fungal cell” refers to any fungal cell that can be transformed with a gene encoding a nicotine biosynthesis enzyme and is capable of expressing in recoverable amounts the enzyme or its products.
  • Illustrative fungal cells include yeast cells such as Saccharomyces cerivisae ( Baldari, et aI., 1987. EMBO J. 6: 229-234 ) and Pichia pastoris (e.g. P. pastoris KM714 available from Invitrogen). Cells of filamentous fungi such as Aspergillus and Trichoderma may also be used.
  • Bacterial cell refers to any bacterial cell that can be transformed with a gene encoding a nicotinic alkaloid biosynthesis enzyme and is capable of expressing in recoverable amounts the enzyme or its products.
  • Illustrative bacterial cells include E. coli, such as E. coli strain M15/rep4, which is available commercially from QIAGEN.
  • Mammalian cell refers to any mammalian cell that can be transformed with a gene encoding a nicotine biosynthesis enzyme and is capable of expressing in recoverable amounts the enzyme or its products.
  • Illustrative mammalian cells include Chinese hamster ovary cells (CHO) or COS cells.
  • Mammalian cells may also include a fertilized oocyte or an embryonic non-human stem cell into which nicotinic alkaloid biosynthesis enzyme-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals. Examples of systems for regulated expression of proteins in mamlian cells include Clontech's Tet-Off and Tet-On gene expression systems and similar systems. Gossen and Bujard, Proc. Natl. Acad. Sci. USA 89: 55475551 (1992 ).
  • Algae cell refers to any algae species that can be transformed with a gene encoding a nicotine biosynthesis enzyme without adversely affecting normal algae development or physiology.
  • Illustrative algae cells include Chlamydomonas reinhardtii ( Mayfield and Franklin, Vaccine 23: 1828-1832 (2005 ).
  • an inducible expression system may mitigate adverse affects. For example, insect cells may be first grown under non-inducing conditions to a desired state and then expression of the enzyme is induced.
  • cells expressing nicotinic alkaloid biosynthesis genes may be supplied with precursors to increase substrate availability for nicotinic alkaloid synthesis.
  • Cells may be supplied with analogs of precursors which may be incorporated into analogs of naturally occurring nicotinic alkaloids.
  • nicotine is the major alkaloid in N. tabacum and some other species in the Nicotiana genus
  • other plants have nicotine-producing ability, including, for example, Duboisia, Anthocericis and Salpiglessis genera in the Solanaceae, and Eclipta and Zinnia genera in the Compositae.
  • nicotine may be produced in non-nicotine producing plants, such as Atropa belladonna and Anabidopsis thaliana, and cells, such as insect, fungal, and bacterial cells.
  • a plant or non-nicotine producing cell such as a fungal cell
  • a plasmid comprising one or more sequences, each operably linked to a promoter.
  • an illustrative vector may comprise a QPT sequence operably linked to a promoter.
  • the plasmid may comprise a QPT sequence operably linked to a promoter and an A622 sequence operably linked to a promoter.
  • a plant or non-nicotine producing cell may be transformed with more than one plasmid.
  • a plant or non-nicotine producing cell may be transformed with a first plasmid comprising a QPT sequence operably linked to a promoter, which is distinct from a second plasmid comprising an A622 or NBB1 sequence.
  • a first plasmid comprising a QPT sequence operably linked to a promoter, which is distinct from a second plasmid comprising an A622 or NBB1 sequence.
  • the first and second plasmids or portions thereof are introduced into the same cell.
  • Transgenic plant refers to a plant that comprises a nucleic acid sequence that also is present per se in another organism or species or that is optimized, relative to host codon usage, from another organism or species. Both monocotyledonous and dicotyledonous angiosperm or gymnosperm plant cells may be transformed in various ways known to the art. For example, see Klein et al., Biotechnology 4: 583-590 (1993 ); Bechtold et al., C. R. Acad. Sci. Paris 316:1194-1199 (1993 ); Bent et al., Mol. Gen. Genet. 204:383-396 (1986 ); Paszowski et al., EMBO J.
  • Agrobacterium species such as A. tumefaciens and A. nhizogenes can be used, for example, in accordance with Nagel et al., Microbiol Lett 67: 325 (1990 ). Additionally, plants may be transformed by Rhizobium, Sinorhizobium or Mesorhizobium transformation. Broothaerts et al., Nature 433:629-633 (2005 ).
  • Agrobacterium may be transformed with a plant expression vector via, e.g., electroporation, after which the Agrobacterium is introduced to plant cells via, e.g ., the well known leaf-disk method. Additional methods for accomplishing this include, but are not limited to, electroporation, particle gun bombardment, calcium phosphate precipitation, and polyethylene glycol fusion, transfer into germinating pollen grains, direct transformation ( Lorz et al., Mol. Genet. 199: 179-182 (1985 )), and other methods known to the art. If a selection marker, such as kanamycin resistance, is employed, it makes it easier to determine which cells have been successfully transformed. Marker genes may be included within pairs of recombination sites recognized by specific recombinases such as cre or flp to facilitate removal of the marker after selection. See U. S. published application No. 2004/0143874 .
  • Transgenic plants without marker genes may be produced using a second plasmid comprising a nucleic acid encoding the marker, distinct from a first plasmid that comprises an A622 or NBB1 sequence.
  • the first and second plasmids or portions thereof are introduced into the same plant cell, such that the selectable marker gene that is transiently expressed, transformed plant cells are identified, and transformed plants are obtained in which the A622 or NBB1 sequence is stably integrated into the genome and the selectable marker gene is not stably integrated. See U. S. published application No. 2003/0221213 .
  • the first plasmid that comprises an A622 or NBB1 sequence may optionally be a binary vector with a T-DNA region that is completely made up of nucleic acid sequences present in wild-type non-transgenic N. tabacum or sexually compatible Nicotiana species.
  • Agrobacterium transformation methods discussed above are known to be useful for transforming dicots. Additionally, de la Pena et al., Nature 325: 274-276 (1987 ), Rhodes et al., Science 240: 204-207 (1988 ), and Shimamato et al., Nature 328: 274-276 (1989 ) have transformed cereal monocots using Agrobacterium. Also see Bechtold et al., C.R. Acad. Sci. Paris 316 (1994 ), illustrating vacuum infiltration for Agrobacterium -mediated transformation.
  • Methods of regenerating a transgenic plant from a transformed cell or culture vary according to the plant species but are based on known methodology. For example, methods for regenerating of transgenic tobacco plants are well-known. Genetically engineered plants are selected that have increased expression of at least one of A622, NBB1 , PMT, and QPT. Additionally, the genetically engineered plants may have increased nicotine levels and yield.
  • the present invention provides methodology for producing nicotine and nicotine analogs, as well as enzymes for synthesis of nicotine and nicotine analogs.
  • These compounds may be produced by genetically engineered nicotine-producing plants and non-nicotine producing cells, as well as in a cell-free/in vitro system.
  • nicotinic alkaloids can be produced in tobacco hairy root culture by expressing at least one of A622 and NBB1.
  • the present disclosure contemplates cell culture systems, such as bacterial or insect cell cultures, for producing large-scale or commercial quantities of nicotine by expressing A622 and NBB1 .
  • the nucleotide sequence of the NBB1 cDNA insert was determined on both strands using an ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems) and a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).
  • the NBB1 full-length cDNA is set forth in SEQ ID NO: 1.
  • the amino acid sequence encoded by the nucleotide sequence is set forth in SEQ ID NO: 2.
  • the protein sequence includes a FAD-binding motif. A putative vacuolar signal peptide is located at the N-terminus.
  • the sequence fragment from 1278 bp through the end (1759 bp) of the NBB1 nucleotide sequence was used as the probe template.
  • the template was prepared by amplification from the cDNA clone by PCR using the following primers:
  • Nectarin V (AFS034411AF503442); HaCHOX, sunflower (Helianthus annuus) carbohydrate oxidase (AF472609); LsCHOX, lettuce (Lactuca sativa) carbohydrate oxidase (AF472608); and 27 Arabidopsis genes (Atlg01980, Atlgl1770, Atlg26380, Atlg26390, Atlg26400, Atlg26410, Atlg26420, Atlg30700, Atlg30710, Atlg30720, Atlg30730, Atlg30740, Atlg30760, Atlg34575, At2g34790, At2g34810, At4g20800, At4g20820, At4g20830, At4g20840, At4g20860, At5g44360, At5g44380, At5g44390, At5g44400, At5g44410, and At5g44440
  • An attB-NBB1 fragment was amplified by PCR using the pGEMTNBB1cDNAfull vector of Example 1 as the template and two sets of primers; one set for the NBB 1 gene-specific amplification (gene-specific primers) and another set to add the attB sequences (adapter primers). PCR conditions were those recommended by the manufacturer.
  • the GATEWAY entry clone pD0NR221-NBB1 was created by a BP recombination reaction between the attB-NBB 1 PCR product and pD0NR221 (Invitrogen).
  • a TobRD2 promoter region (SEQ ID NO: 5 in WO9705261 ) of 1,031 bp was amplified using Burley 21 genomic DNA as the template and the TobRD2 promoter-specific primers, and then digested with HindIII and XbaI.
  • the NBB1 ORF was transferred by an LR reaction from the pDONR221-NBB1 vector to a GATEWAY binary vector pTobRD2-DEST, which was designed to express a cloned ORF under the TobRD2 promoter.
  • pTobRD2-NBB1ox A diagram of the T-DNA region of the final expression vector, pTobRD2-NBB1ox, is shown in Figure 4B .
  • the binary vector pTobRD2-NBB1 ox was introduced to Agrobacterium rhizogenes strain 15834 by electroporation. Nicotiana tabacum cv. K326 wild-type plants were transformed by A. rhizogenes using a leaf-disc method, basically as described by Kanegae et al., Plant Physiol. 105:2:483-90 (1994 ). Kanamycin resistance (200 mg/L in B5 medium) was used as a selection marker for the pTobRD2-NBB1ox. transformed lines (TN lines). Wild-type A. rhizogenes was used to produce control hairy root lines (WT lines). Tobacco hairy roots were grown in the B5 liquid medium at 27 °C under the dark condition for two weeks, and then harvested.
  • NBB1 protein Expression levels of the NBB1 protein were analyzed by an immunoblot analysis. Hairy roots were frozen in liquid nitrogen, and immediately homogenized using a mortar and pestle in an extraction buffer (100 mM Tris-HCl pH6.8, 4% SDS, 20% glycerol) containing 1mM phenylmethylsulfonyl fluoride and 200 mM dithiothreitol. After centrifugation of the homogenates, soluble proteins in the supernatant were separated by SDS-PAGE. Immunoblot analysis was performed using an anti-NBB1 rabbit serum. The detailed procedures were reported previously. Shoji et al., Plant Mol. Biol., 50, 427-440 (2002 ). Immunoblots with anti-NBB1 antiserum show that the transgenic hairy root lines TN9 and TN17 have increased levels of NBB1 protein. See Figure 5A .
  • Transgenic hairy roots were cultured for two weeks, collected, and freeze dried. 2 ml of 0.1 N sulfuric acid was added to 19 mg of the freeze-dried sample. This suspension was sonicated for 15 minutes, and filtered. Ammonium hydroxide (0.1 ml, 28% NH 3 ; Wako) was added to 1 ml of the filtrate, and the mixture was centrifuged for 10 minutes at 15,000 rpm. A sample of the supernatant (1 ml) was loaded onto an Extrelut-1 column (Merck) and let sit for 5 minutes. Alkaloids were eluted with 6 ml of chloroform.
  • Ammonium hydroxide 0.1 ml, 28% NH 3 ; Wako
  • the eluted chloroform fraction was then dried under reduced pressure at 37°C with an evaporator (Taitec Concentrator TC-8).
  • the dried sample was dissolved in 50 ⁇ l of ethanol solution containing 0.1 % dodecane.
  • a gas chromatography apparatus (GC-14B, Shimadzu) equipped with a capillary column (Rtx-5Amine column, Restec) and an FID detector was used to analyze the samples.
  • the column temperature was maintained at 100 °C for 10 min, elevated to 150 °C at 25 °C/min, held at 150 °C for 1 min, elevated to 170 °C at 1 °C/min, held at 170 °C for 2 min, elevated to 300 °C at 30 °C/min, and then held at 300 °C for 10 min.
  • Injection and detector temperature was 300 °C.
  • a 1 ⁇ l sample of the purified alkaloid preparation was injected, and alkaloid contents were measured by the internal standard method.
  • AttB-A622 fragment was amplified using the pcDNAII-A622 vector, per Hibi et al, Plant Cell 6: 723-35 (1994 ), as the template, the A622-specific primers below, and adapter primers, as described above for Example 4.
  • the amplified A622 fragment was cloned into the pD0NR221 vector by BP reaction, resulting in pDONR-A622, and then the A622 fragment was transferred from pDONR-A622 to pTobRD2-DEST by an LR reaction.
  • the resultant expression vector was referred to as pTobRD2-A622ox.
  • a diagram of the T-DNA region of pTobRD2-A622ox is shown in Figure 4C .
  • N. tabacum cv. K326 wild-type plants were transformed by A. rhizogenes 15834 containing the pTobRD2-A622ox vector, as described above for Example 4.
  • Transgenic hairy roots carrying the T-DNA from pTobRD2-A622ox were referred to as TA lines, and cultured as described above in Example 4.
  • the TobRD2-A622 expression cassette and the TobRD2-NBB1 expression cassette were cloned in tandem in a binary vector.
  • the TobRD2-A622 cassette was cut from pTobRD2-A622ox with HindIII, and then cloned into the HindIII site at the 5' end of the TobRD2 promoter in pTobRD2-NBB1ox.
  • the resultant vector for overexpression of both NBB1 and A622 was referred to as pTobRD2-A622ox-NBB1ox.
  • a diagram of the T-DNA region of pTobRD2-A622ox-NBB1ox is shown in Figure 4D .
  • Nicotiana tabacum cv. K326 wild-type plants were transformed by A. rhizogenes 15834 containing the pTobRD2-A622ox-NBB1ox vector, as described above for Example 4.
  • Transgenic hairy roots carrying the T-DNA from pTobRD2-A622ox-NBB1ox were referred to as TNA lines, and cultured as described above in Example 4.
  • EXAMPLE 7 Transgenic A. belladonna plants expressing A622 protein
  • Atropa belladonna produces tropane alkaloids, hyoscyamine and scopolamine, which are derived from the N-methylpyrrolinium cation, but does not contain nicotine alkaloids, possibly due to the absence of NBB 1 and A622 genes.
  • Tobacco A622 cDNA containing an introduced NcoI site at the first ATG was excised from the pcDNAII-A622 vector (Hibi et al. 1994) as an NcoI-BamHI fragment and cloned into pRTL2 ( Restrepo et al., Plant Cell 2:987-98 (1990 )) under the control of a CaMV35S promoter with a duplicated enhancer.
  • This A622 overexpression cassette was excised with HindIII and cloned in a binary vector pGA482 (Amersham) to produce the A622 expression vector pGA-A622.
  • the binary vector pGA-A622 was introduced to A. tumefaciens strain EHA105 by electroporation.
  • A. belladonna plants were transformed by A. tumefaciens using a leaf-disk method, basically as described by Kanegae et al., (Plant Physiol. 105(2):483-90 (1994 )).
  • Kanamycin resistance 200 mg/L in MS/B5 medium
  • Transgenic 35S-A622 plants were regenerated from the leaf discs, grown at 22 °C under continuous light in a growth chamber.
  • Total proteins were extracted from leaves of wild-type and 35S-A622 T1 plants, as described above in Example 5. Immunoblot analysis was performed using anti-A622 mouse serum. Leaf tissues of the self-pollinated T1 generation plants that contained high amounts of A622 protein, such as line C1#3 (see Figure 7 ), were used for alkaloid analysis.
  • Nicotinic alkaloids in transgenic A. belladonna plants were extracted with 1M H 2 SO 4 and purified basically as described (Hashimoto et al., 1992). Alkaloids were identified by gas chromatography-mass spectrometry (GC-MS) (Hewlett Packard 5890 series II/JEOL MStation JMS700 with HP-5ms column) after comparison of their mass spectra to those of authentic standards.
  • GC-MS gas chromatography-mass spectrometry
  • the column temperature was maintained at 100 °C for 10 min, elevated to 150 °C at 25 °C/min, held at 150 °C for 1 min, elevated to 170 °C at 1 °C/min, held at 170 °C for 2 min, elevated to 300 °C at 30 °C/min, and then held at 300 °C for 10 min.
  • Introduction of A622 alone in A. belladonna did not result in accumulation of nicotine or other nicotine alkaloids.
  • EXAMPLE 8 Transgenic A. belladonna hairy roots expressing NBB1 protein and
  • transgenic A. belladonna hairy roots that express both A622 and NBB1 by transforming leaves of the A622-expressing Atropa plants of Example 7 with A. rhizogenes strain 15834, which carries an NBB1 expression vector.
  • the binary vector pBE2113 carrying CaMV35S promoter with a duplicated enhancer (El2) and 5'-upstream sequence of tobacco mosaic virus ( ⁇ ) was obtained from Dr. Yuko Ohashi, National Institute of Agrobiological Resources (Tsukuba, Japan), see Plant Cell Physiol. 37: 49-59 (1996 ), and was converted into a GATEWAY destination vector after the GATEWAY cassette, containing att R recombination sites flanking a ccd B gene and a chloramphenicol-resistance gene, was cloned between the XbaI and SacI sites in the vector, which replaced the ⁇ -glucronidase gene with the GATEWAY cassette.
  • the resultant destination binary vector was digested with HindIII and SacI, and the HindIII-SacI fragment containing the El2-35S-S2-GATEWAY cassette was cloned between the HindIII and SacI sites in pBI101H.
  • the resultant destination binary vector was referred to as pBI101H-E2113-DEST.
  • a diagram of the T-DNA region of pBI101H-E2113-DEST is shown in Figure 4E .
  • NBB1 ORF was transferred from the pDONR221-NBB1-2 vector to the GATEWAY binary vector pBI101H-E2113-DEST by an LR reaction.
  • the expression vector was referred to as pEl235S ⁇ -NBB1.
  • a diagram of the T-DNA region of pE1235S ⁇ -NBB1 is shown in Figure 4F .
  • the transgenic A. belladonna E2 and WT hairy roots were cultured for 3 weeks in 100 ml of MS/B5 liquid medium containing 100mg/l nicotinic acid. Nicotine alkaloids in E2 and WT hairy roots were extracted with 1M H 2 SO 4 and purified basically as described (Hashimoto et al., 1992). Alkaloids were identified by gas chromatography-mass spectrometry (GC-MS) (Hewlett Packard 5890 series II/JEOL MStation JMS700 with HP-5ms column) after comparison of their mass spectra to those of authentic standards.
  • GC-MS gas chromatography-mass spectrometry
  • the column temperature was maintained at 100°C for 10 min, elevated to 150°C at 25°C/minute, held at 150°C for 1 minute, elevated to 170 °C at 1°C/min, held at 170°C for 2 minutes, elevated to 300 °C at 30°C/min, and then held at 300°C for 10 minutes.
  • a small but distinct novel peak was detected (See peak 5 in Figure 9 ).
  • a peak corresponding to the peak 5 was not detectable in the WT line hairy roots.
  • the compound of peak 5 showed an MS fragmentation profile identical to that of nicotine, as shown in Figure 10 . This demonstrated that expression of exogenous NBB 1 and A622 are sufficient for nicotine formation in A. belladonna hairy roots.
  • COMPARATIVE EXAMPLE 9 Increasing nicotine content by expression of PMT under control of the A622 promoter
  • pA622pro-DEST has the NPTII gene expression cassette and the HPT gene expression cassette as selection markers.
  • An A622 promoter of 1,407 bp was amplified using a vector containing the A622 promoter ( Shoji et aI., Plant Mol. BioI. 50,427-440 (2002 )) as the template and the A622 promoter-specific p ⁇ imers shown below, and digested with HindIII and Xbal. The resultant fragment was cloned between the HindIII and XbaI sites in pBIlOIH.
  • AttB-PMT fragment was amplified using the tobacco PMT vector in which PMT ORF (NCBI accession number; D28506) was cloned in the BstXI site of pcDNAII (Invitrogen) (See SEQ ID NO: 7B) as the template, the gene-specific primers below, and attB sequence adapter primers, as described above in Example 4.
  • a GATEWAY entry clone pDONR221-PMT was created by a BP recombination reaction between the attB-PMT PCR product and pD0NR221 (Invitrogen).
  • the PMT ORF was transferred from the pDONR221-PMT vector to the GATEWAY binary vector pA622pro-DEST by an LR reaction.
  • the gene expression vector was referred to as pA622pro-PMTox. See Figure 11B .
  • pA622pro-PMTox was transformed into Agrobacteriuin tumefaciens strain EHA105, which was used to transform wild-type K326 leaves.
  • Transgenic T0 shoots were regenerated, and were transferred to the rooting medium. Several rooted transgenic plants were transferred to soil.
  • Alkaloids were extracted from the transgenic tobacco leaves and analyzed, as described above in Example 4. The nicotine content in leaves of plants sampled 36 days after transfer to soil was analyzed. Several transgenic lines transformed with pA622pro-PMTox showed greater nicotine accumulation than the control lines, in which wild-type K326 plants were transformed with the AG-GUS cassette. See Figure 12 .
  • COMPARATIVE EXAMPLE 10 Increasing nicotine content by expression of PMT under control of the TobRD2 promoter
  • the PMT ORF was transferred from the pD0NR22I-PMT vector to the GATEWAY binary vector pTobRD2-DEST (see Figure 4A ) by an LR reaction.
  • the gene expression vector was referred to as pTobRD2-PMTox. See Figure 11 C .
  • pTobRD2-PMTox was transformed into Agrobacterium tumefaciens strain ERAI05, which was used to transform wild-type K326 leaves.
  • Transgenic TO shoots were regenerated, and were transferred to the rooting medium. Several rooted transgenic plants were transferred to soil.
  • Alkaloids were extracted from the transgenic tobacco leaves and analyzed, as described above in Example 4. The nicotine content in leaves of plants sampled 36 days after transfer to soil was analyzed. Several transgenic lines showed greater nicotine accumulation than the control lines, in which wild-type K326 plants were transformed with a TobRD2-GUS cassette. See Figure 13 .
  • COMPARATIVE EXAMPLE 11 Increasing nicotine content by expression of QPT under control of the A622 promoter
  • the QPT ORF fragment (SEQ ID NO: 5B) was amplified using the pBJY6 vector (supplied from Dr. Kenzo Nakamura, Nagoya University, Japan) as the template and the gene-specific primers shown below.
  • a GATEWAY entry clone pENTR-QPT was created by a TOPO cloning reaction.
  • the QPT ORF was transferred from the pENTR-QPT vector to the GATEWAY binary vector pA622pro-DEST (see Figure 11A ) by an LR reaction.
  • the gene expression vector was referred to as pA622pro-QPTox. See Figure 11D .
  • Alkaloids were extracted from the transgenic tobacco leaves and analyzed, as described above in Example 4. The nicotine content in leaves of plants sampled 36 days after transfer to soil was analyzed. Several transgenic lines showed greater nicotine accumulation than the control lines, in which wild-type K326 plants were transformed with an A622-GUS cassette. See Figure 14 .
  • COMPARATIVE EXAMPLE 12 Increasing nicotine content by expression of QPT under control of the TobRD2 promoter
  • the QPT ORF was transferred from the pD0NR221-QPT vector to a GATEWAY binary vector pTobRD2-DEST (see Figure 4A ) by an LR reaction.
  • the gene expression vector was referred to as pTobRD2-QPTox. See Figure 11E .
  • pTobRD2-QPTox was transformed into Agrobacterium tumefaciens strain EHA105, which was used to transform wild-type K326 leaves.
  • Transgenic T0 shoots were regenerated, and were transferred to the rooting medium. Several rooted transgenic plants were transferred to soil.
  • Alkaloids were extracted from the transgenic tobacco leaves and analyzed, as described above in Example 4. The nicotine content in leaves of plants sampled 36 days after transfer to soil was analyzed. Several transgenic lines showed greater nicotine accumulation than the control lines, in which wild-type K326 plants were transformed with a TobRD2 -GUS cassette. See Figure 15 .
  • COMPARATIVE EXAMPLE 13 Increasing nicotine content by expression of PMT and QPT under control of the A622 promoter
  • the vector was converted into a GATEWAY destination vector after the GATEW A Y cassette containing att R recombination sites flanking a ccd B gene and a chloramphenicol-resistance gene was cloned between the XbaI and SacI sites in the vector, which replaced the B -glucronidase gene with the GATEWAY cassette. Then, an HindIII-EcoRI adapter was inserted into the EcoRI site at the 3' end of Nos terminator resulting in pBI221-A622pro-DEST.
  • the A622pro-PMT expression cassette and the A622pro-QPT expression cassette were cloned in tandem in a binary vector.
  • the PMT ORF was transferred from the pDONR221-PMT vector to the GATEWAY binary vector pBI221-A622pro-DEST by an LR reaction.
  • the gene expression vector was referred to as pBI221-A622pro-PMT.
  • the pBI221-A622proPMT was digested with HindIII, and then cloned into the HindIII site at the 5' end of A622 promoter of the pA622pro-QPTox vector.
  • the resultant PMT-QPT expression vector was referred to as pA622pro-PMTox-QPTox.
  • a diagram of the T-DNA region of pA622pro-PMTox-QPTox is shown in Figure 11F .
  • pA622pro-PMTox-QPTox was transformed into Agrobacterium tumefaciens strain ERA 1OS, which was used to transform wild-type K326 leaves.
  • Transgenic TO shoots were regenerated, and were transferred to the rooting medium. Several rooted transgenic plants were transferred to soil.
  • Alkaloids were extracted from the transgenic tobacco leaves and analyzed, as described above in Example 4. The nicotine content in leaves of plants sampled 36 days after transfer to soil was analyzed. Several lines transformed with A622pro-PMToxQPTox showed greater nicotine accumulation than the control lines, in which wild-type K326 plants were transformed with an A622-GUS cassette. See Figure 16 .
  • COMPARATIVE EXAMPLE 14 Increasing nicotine content by expression of PMT and QPT under control of the TobRD2 promoter
  • pTobRD2-PMTox-QPTox was transformed into Agrobacterium tumefaciens strain EHA105, which was used to transform wild-type K326 leaves.
  • Transgenic TO shoots were regenerated, and were transferred to the rooting medium. Several rooted transgenic plants were transferred to soil.
  • the binary vector pGA-A622 was introduced to A. tumefaciens strain LBA4404 by electroporation.
  • A. thaliana plants ecotype: Wassilewskija (WS)
  • WS Wassilewskija
  • Kanamycin resistance 50 mg/L on Shoot-Induction medium
  • Transgenic plants were regenerated from the callus, grown at 23 °C under 16h light/8h dark condition in a growth chamber.
  • NBB1 ORF (SEQ ID NO: 1B)was transferred from the pDONR221-NBB1-2 vector to a GATEWAY binary vector pGWB2 (see Figure 18A ) by LR reaction.
  • the gene expression vector, in which NBB1 is linked to the CAMV 35S promoter, is referred to as p35S -NBB1.
  • PMT ORF was transferred from the pDONR221-PMT vector to the pGWB2 by LR reaction.
  • the gene expression vector is referred to as p35S -PMT. See Figure 18C .
  • the binary vectors p35S- NBB1 and p35S- PMT were introduced to A. tumefaciens strain EHA105 by electroporation.
  • T1 generation plants carrying pGA- A622 were transformed by A. tumefaciens using a floral dip method, basically as described by Clough et al., Plant J. 16: 735-43 (1998 ).
  • Hygromycin resistance 25 mg/L on Shoot-Induction medium
  • Transgenic plants were grown at 23 °C under 16h light/8h dark condition in a growth chamber. Resultant Transgenic plants were screened by genomic PCR using the 35S promoter primers and NBB1- or PMT- gene specific primers.
  • the PCR positive 35S- A622- 35S -NBB1 plants and 35S- A622- 35S -PMT plants were crossed to produce 35S -A622- 35S -NBB1- 35S -PMT plants.
  • F1 progeny were screened by genomic PCR using each expression cassette specific primer pair.
  • NBB1 and A622 ORFs SEQ ID NO: 1B and 3B, respectively
  • DONR vectors DONR-NBB1-2, pDONR-A622
  • GATEWAY vector pDEST10 Invitrogen
  • NBB 1 and A622 were produced in the insect cell cultures, as shown by immunoblotting with anti-NBB1 and anti-A622 antisera.
  • the recombinant proteins containing the 6xHis tag were purified by adsorption on Ni-NTA columns followed by elution with 0.5 M imidazole. See Figure 21B .

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  • Heart & Thoracic Surgery (AREA)
  • Botany (AREA)
  • Hospice & Palliative Care (AREA)

Claims (7)

  1. Procédé d'augmentation de la teneur en nicotine d'une plante Nicotiana, comprenant la surexpression du gène A622 par rapport à une plante témoin.
  2. Procédé selon la revendication 1, comprenant en outre une surexpression du gène NBB1.
  3. Procédé selon la revendication 2, comprenant en outre une surexpression d'au moins l'un des gènes QPT et PMT, préférablement dans lequel le gène QPT et le gène A622 sont surexprimés.
  4. Procédé d'augmentation de la teneur en nicotine d'une plante Nicotiana, comprenant :
    (a) la transformation d'une plante Nicotiana avec une construction comprenant, dans la direction 5' à 3', un promoteur fonctionnellement lié à un acide nucléique hétérologue codant pour une enzyme qui augmente la synthèse de la nicotine, ledit acide nucléique étant le gène A622 et le gène A622 étant surexprimé ;
    (b) la régénération de plantes Nicotiana transgéniques à partir de la plante transformée ; et
    (c) la sélection d'une plante Nicotiana transgénique présentant une teneur en nicotine augmentée par rapport à une plante témoin.
  5. Procédé d'augmentation de la teneur en nicotine et du rendement d'une plante Nicotiana selon la revendication 1, comprenant :
    (a) la transformation d'une plante Nicotiana avec (i) une première construction comprenant, dans la direction 5' à 3', un promoteur fonctionnellement lié à un acide nucléique hétérologue codant pour une enzyme qui augmente la synthèse de la nicotine ; et (ii) une deuxième construction comprenant, dans la direction 5' à 3', un promoteur fonctionnellement lié à un acide nucléique hétérologue codant pour une enzyme qui augmente le rendement, ladite première construction comprenant un acide nucléique codant pour l'enzyme A622 et le gène A622 étant surexprimé ;
    (b) la régénération de plantes Nicotiana transgéniques à partir de la plante transformée ; et
    (c) la sélection d'une plante Nicotiana transgénique présentant une teneur en nicotine augmentée et un rendement augmenté par rapport à une plante témoin.
  6. Plante Nicotiana produite par l'un quelconque des procédés selon les revendications 1 à 5, ladite plante surexprimant le gène A622 et présentant une teneur en nicotine augmentée par rapport à une plante témoin.
  7. Utilisation de la plante selon la revendication 6, pour produire un produit sélectionné dans le groupe constitué d'une cigarette, d'un produit pharmaceutique, ou d'un produit nutriceutique.
EP06848676.0A 2006-09-13 2006-09-13 Augmentation des niveaux d'alcaloides nicotiniques Active EP2061889B1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
EP19177939.6A EP3578661A1 (fr) 2006-09-13 2006-09-13 Augmentation des taux d'alcaloïdes nicotiniques
EP20110187201 EP2450446B1 (fr) 2006-09-13 2006-09-13 Augmentation des niveaux d'alcaloïdes nicotiniques
EP14163682.9A EP2792750B1 (fr) 2006-09-13 2006-09-13 Augmentation des taux d'alcaloïdes nicotiniques
PL14163682T PL2792750T3 (pl) 2006-09-13 2006-09-13 Zwiększanie poziomów alkaloidów nikotynowych
PL11187201T PL2450446T3 (pl) 2006-09-13 2006-09-13 Zwiększanie poziomów alkaloidów nikotynowych
PL06848676T PL2061889T3 (pl) 2006-09-13 2006-09-13 Zwiększanie poziomów alkaloidów nikotynowych
HK15103733.9A HK1203545A1 (en) 2006-09-13 2015-04-16 Increasing levels of nicotinic alkaloids

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IB2006/004043 WO2007072224A2 (fr) 2006-09-13 2006-09-13 Augmentation des niveaux d'alcaloides nicotiniques

Related Child Applications (4)

Application Number Title Priority Date Filing Date
EP19177939.6A Division EP3578661A1 (fr) 2006-09-13 2006-09-13 Augmentation des taux d'alcaloïdes nicotiniques
EP14163682.9A Division EP2792750B1 (fr) 2006-09-13 2006-09-13 Augmentation des taux d'alcaloïdes nicotiniques
EP20110187201 Division EP2450446B1 (fr) 2006-09-13 2006-09-13 Augmentation des niveaux d'alcaloïdes nicotiniques
EP20110187201 Division-Into EP2450446B1 (fr) 2006-09-13 2006-09-13 Augmentation des niveaux d'alcaloïdes nicotiniques

Publications (2)

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EP2061889A2 EP2061889A2 (fr) 2009-05-27
EP2061889B1 true EP2061889B1 (fr) 2014-04-23

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EP20110187201 Not-in-force EP2450446B1 (fr) 2006-09-13 2006-09-13 Augmentation des niveaux d'alcaloïdes nicotiniques
EP06848676.0A Active EP2061889B1 (fr) 2006-09-13 2006-09-13 Augmentation des niveaux d'alcaloides nicotiniques
EP19177939.6A Withdrawn EP3578661A1 (fr) 2006-09-13 2006-09-13 Augmentation des taux d'alcaloïdes nicotiniques
EP14163682.9A Active EP2792750B1 (fr) 2006-09-13 2006-09-13 Augmentation des taux d'alcaloïdes nicotiniques

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EP19177939.6A Withdrawn EP3578661A1 (fr) 2006-09-13 2006-09-13 Augmentation des taux d'alcaloïdes nicotiniques
EP14163682.9A Active EP2792750B1 (fr) 2006-09-13 2006-09-13 Augmentation des taux d'alcaloïdes nicotiniques

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EP (4) EP2450446B1 (fr)
JP (1) JP5087777B2 (fr)
ES (3) ES2748823T3 (fr)
HK (2) HK1169677A1 (fr)
HU (1) HUE026819T2 (fr)
PL (3) PL2792750T3 (fr)
PT (1) PT2450446E (fr)
TW (4) TWI659102B (fr)
WO (1) WO2007072224A2 (fr)

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TW201925466A (zh) 2019-07-01
EP2450446B1 (fr) 2015-04-29
ES2748823T3 (es) 2020-03-18
TWI421341B (zh) 2014-01-01
HK1169677A1 (en) 2013-02-01
JP2010505449A (ja) 2010-02-25
JP5087777B2 (ja) 2012-12-05
TW201623611A (zh) 2016-07-01
EP2792750A1 (fr) 2014-10-22
PL2061889T3 (pl) 2014-11-28
WO2007072224A3 (fr) 2008-04-17
HUE026819T2 (en) 2016-07-28
EP2450446A2 (fr) 2012-05-09
TW201414834A (zh) 2014-04-16
TWI659102B (zh) 2019-05-11
EP2792750B1 (fr) 2019-07-03
ES2543907T3 (es) 2015-08-25
PL2792750T3 (pl) 2020-02-28
PL2450446T3 (pl) 2015-10-30
PT2450446E (pt) 2015-09-15
ES2478633T3 (es) 2014-07-22
HK1203545A1 (en) 2015-10-30
EP2450446A3 (fr) 2012-07-11
WO2007072224A2 (fr) 2007-06-28
TW200817509A (en) 2008-04-16
TWI657141B (zh) 2019-04-21
EP3578661A1 (fr) 2019-12-11
EP2061889A2 (fr) 2009-05-27
WO2007072224A8 (fr) 2009-04-23

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